Abstract: :
Purpose: The aim of this study was to determine the differentiationcharacteristics of a previously established telomerase immortalizedhuman corneal epithelial cell (THC) line compared with normalhuman corneal epithelium.Methods: THC were grown on cell cultureinserts (12mm diameter, 3.0µm pore size, Corning Inc.,Corning, NY) submersed in high calcium containing (1.15mmol/L)KGM-2 media (CloneticsTM, BioWhittaker, Inc., Walkersville,MD) for seven days. THC cultures were then air-lifted and evaluatedat day 0, 7 and 10 days by TdT-mediated dUTP-biotin nick endlabeling (TUNEL, ApopTag®, Serologicals Corporation, Norcross,GA) and immunohistochemistry (IHC) using antibodies specificfor Muc1, Bcl-2 and Keratin type 3 (K3). Expression of proteinswas confirmed by Western Blotting. TUNEL and IHC image wereobtained by either Leica SPII laser confocal or fluorescencemicroscopy with CoolSNAPfxTM CCD camera (Roper Scientific, Tucson,AZ).Results: After 7 days of submersed culture (day 0), THCformed a confluent layer, one to two cells thick. After air-liftingfor 7 days (day 7), there was clear stratification of the epithelialsheet to form a cuboidal basal cell layer, a polygonal wingcell layer (1-2 cells) and a squamous superficial cell layer(1-2 cells). At 10 days occasional intraepithelial cysts appearedin the basal/wing cell layer. Similar to normal cornea, anti-Bcl-2Mabs to the aa 11-44 epitope stained the nucleus of HTC. Interestingly,occasional superficial epithelial cell nuclei failed to stainwith anti-Bcl-2 by day 7 and 10, again similar to differentiatednormal human and rabbit corneas. TUNEL positive cells were notdetected at day 0, but by day 7 and 10 occasional TUNEL labelingwas detected in the superficial epithelial cells. Importantly,superficial TUNEL positive cells were negative for nuclear Bcl-2,again similar to normal cornea. Muc1 localized to cytoplasmicmembrane in all cells at day 0; however, by days 7 to 10 Muc1localized only to superficial epithelial cells and on occasionalbasal cell. K3 showed staining in the cytoplasm of THC at day0, but was enhanced in upper layer and reduced in basal layerat days 7 to 10.Conclusions: Overall these data show thatcultured-THC exhibit natural human/rabbit epithelial differentiationpattern after 7-days of air-lifting. Importantly, differentiationin culture appears to mimic normal programmed cell death ofsurface cells with loss of Bcl-2 prior to apoptosis.